Systems and Methods to Treat Cardiac Pacing Conditions

ABSTRACT

The method may include administering to a subject in need thereof an effective amount of an HCN polynucleotide. The HCN polynucleotide includes a nucleotide sequence encoding an HCN polypeptide having channel activity. The amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, where the reference polypeptide begins with an amino acid selected from amino acids 92-214 and ends with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8. An example of a reference polypeptide is amino acids 214-723 of SEQ ID NO:8. The HCN polynucleotide may be DNA or RNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/351,836 filed Jun. 4, 2010 and cross reference is hereby made to thecommonly assigned related U.S. application Ser. No. ______ (AttorneyDocket Number P0036355.02), entitled “Compositions and Methods to TreatCardiac Pacing Conditions” and U.S. application Ser. No. ______(Attorney Docket Number P0036355.04), entitled “Compositions to TreatCardiac Pacing Conditions), filed concurrently herewith and incorporatedherein by reference in its entirety.

BACKGROUND

Cardiac contraction in a healthy human heart is initiated by spontaneousexcitation of the sinoatrial (“SA”) node, which is located in the rightatrium. The electric impulse generated by the SA node travels to theatrioventricular (“AV”) node where it is transmitted to the bundle ofHis and to the Purkinje network. The fibers in the Purkinje networkbranch out in many directions to facilitate coordinated contraction ofthe left and right ventricles. In some disease states, the heart losessome of its natural capacity to pace properly. Such dysfunction iscommonly treated by implanting a pacemaker.

While effectively improving the lives of many patients, implantablepacemakers have certain technical limitations. For example, implantablepacemakers rely on a self-contained power source such as a battery andconsequently have a limited lifetime before the power source is in needof replacement. Implantable pacemakers also require pacing leads, whichmay fail and result in loss of therapy. Hence, an otherwise healthypatient may require multiple surgeries to replace the power source,leads, or the entire implantable pacemaker. Also, implantable pacemakersmay not directly respond to physiological signals similar to the way theSA node responds to such signals.

Recently, biological methods of influencing a patient's cardiac cellshave been developed, some of which include administeringbiopharmaceutical compositions that affect cardiac pacing. Developmentsin genetic engineering have produced methods for genetically modifyingcardiac cells to modify non-pacemaking cardiac cells to pacemaker-likecardiac cells or regenerate the pacing capabilities of cells in theconduction system of the heart. For example, Johns and Marban (U.S. Pat.No. 6,214,620) describes a method for modulating the excitability ofventricular cells by controlling the regulation of the expression ofcertain ion channels (e.g. K⁺ channels). Marban and Li (PCT PublicationNo. WO 02/087419) and Sigg et al. (PCT Publication No. WO 05/062890A3)describe methods and systems for modulating electrophysiologicalbehavior of cardiac cells by genetic modification of inwardly rectifyingK⁺ channels (I_(K1)) in quiescent ventricular cells.

Another recent biological approach for modulating cardiac pacinginvolves implanting into the SA node or other suitable heart regionscells having particular ion channels that are commonly referred to ashyperpolarization-activated and cyclic nucleotide-gated (HCN) channels.For example, see Rosen and Robinson (PCT Publication No. WO 02/098286)and Sigg et al. (PCT Publication No. WO 05/062958A2). Physiologicallyoriginating in the SA node, the HCN channels play a prominent role inthe control of rhythmic electrical heart activity. Cyclic nucleotidesmodulate the HCN channel activity, and channel activation occurs uponhyperpolarization rather than depolarization. There are four isoforms ofHCN channels (HCN1-4), and each has greater or lesser prevalence indifferent heart regions. Because the HCN isoforms are directly involvedin pacemaker current modulation and activation, implantation ofHCN-expressing cells into cardiac tissue that is diseased orexperiencing conduction blockage is a viable method for regulatingcardiac pacemaker function.

SUMMARY OF THE INVENTION

The present invention provides methods for treating a cardiac pacingcondition. The method may include administering to a subject in needthereof an effective amount of an HCN polynucleotide. The HCNpolynucleotide includes a nucleotide sequence encoding an HCNpolypeptide having channel activity. The amino acid sequence of the HCNpolypeptide and the amino acid sequence of a reference polypeptide haveat least 80% identity, where the reference polypeptide begins with anamino acid selected from amino acids 92-214 and ends with an amino acidselected from amino acids 723-1188 of SEQ ID NO:8. An example of areference polypeptide is amino acids 214-723 of SEQ ID NO:8. The HCNpolynucleotide may be DNA or RNA. The HCN polynucleotide may be presentin a vector, such as a viral vector (including, for instance, a singlestrand adeno-associated virus or a self complementary adeno-associatedvirus), a transposon vector, or a plasmid vector.

The HCN polynucleotide administered to the subject may be present in agenetically modified cell, and the HCN polynucleotide may be integratedin the genomic DNA of the genetically modified cell, or may be presentas part of an extra-chromosomal vector in the cell. The method mayfurther include administering to the subject a second HCNpolynucleotide.

The HCN polynucleotide may be administered by introduction of the HCNpolynucleotide into cardiac atrium cells or cardiac ventricle cells.Various methods may be used to introduce the HCN polynucleotide,including, for instance, a syringe or a catheter.

The present invention also provides a composition that includes an HCNpolynucleotide present in a vector. Preferably, the HCN polynucleotideincludes a nucleotide sequence encoding an HCN polypeptide havingchannel activity, wherein the amino acid sequence of the HCN polypeptideand the amino acid sequence of a reference polypeptide have at least 80%identity, and where the reference polypeptide begins with an amino acidselected from amino acids 92-214 and ends with an amino acid selectedfrom amino acids 723-1188 of SEQ ID NO:8. The composition may furtherinclude a pharmaceutically acceptable carrier. The vector may be a viralvector (including, for instance, a single strand adeno-associated virusor a self complementary adeno-associated virus), a transposon vector, ora plasmid vector.

Further provided by the invention is a genetically modified cell thatincludes an HCN polynucleotide. Preferably, the HCN polynucleotideincludes a nucleotide sequence encoding an HCN polypeptide havingchannel activity, wherein the amino acid sequence of the HCN polypeptideand the amino acid sequence of a reference polypeptide have at least 80%identity, and where the reference polypeptide begins with an amino acidselected from amino acids 92-214 and ends with an amino acid selectedfrom amino acids 723-1188 of SEQ ID NO:8. The HCN polynucleotide may beintegrated in the genomic DNA of the genetically modified cell, or maybe present as part of an extra-chromosomal vector in the cell. Thegenetically modified cell may be part of a composition, and thecomposition may include a pharmaceutically acceptable carrier.

The present invention is also directed to methods that includeidentifying a distal end of a catheter at a tissue site of a patient anddelivering a fluid or polymer that contains an HCN polynucleotide to thetissue site of the patient via the catheter. The identifying may includean electrically sensing contact between a distal end of a catheter andthe tissue site of the patient.

The method may further include delivering an electrical stimulus to thecardiac tissue site of the patient to enhance transfer of the HCNpolynucleotide to the tissue site via electroporation. The electricalstimulus may be delivered to the tissue site via the catheter, forinstance, via an electrode coupled to the catheter and an electrodecoupled to a distal tip of a probe extending from the catheter. Anexample of delivering the electrical stimulus to the tissue site via thecatheter may include delivering the electrical stimulus to the tissuesite via an electrode coupled to the catheter and a distal tip of aprobe extending from the catheter, the distal tip of the probe is formedfrom an electrically conductive material. The electrical stimulus may bedelivered to the tissue site via an implanted medical device. Theelectrical stimulus delivered to the tissue site may include astimulation pulse, or a series of stimulation pulses. The deliveringfluid to the tissue site of the patient via the catheter may includedelivering fluid to the tissue site of the patient via one or more exitports of a distal tip of a probe extending from the catheter. The distaltip of the probe may include a needle or may be a helix shaped distaltip. The distal tip of the probe may extend from a body of the catheterupon sensing contact between the tissue site of the patient and thecatheter.

Also provided by the present invention is a system that includes a fluidsupply, a catheter, and a power supply to generate an electricalstimulus that is delivered to the tissue site. The system may include apump to drive fluid from the fluid supply through the catheter, and/orthe power supply may include an implanted medical device that deliversthe electrical stimulus to the tissue site. The implanted medical devicemay include one of an implantable pulse generator, an implantablecardioverter/defibrillator, and an implantablepacemaker/cardioverter/defibrillator. The fluid supply may include animplanted fluid reservoir. The power supply may be coupled to thecatheter, and the catheter may deliver the electrical stimulus to thetissue site.

The catheter of the system may include a catheter body that defines aninner lumen, a probe within the inner lumen that delivers fluid from thefluid supply to a tissue site of a patient, and at least one electrodecoupled to the catheter to detect contact between the catheter and thetissue site, wherein the fluid comprises an HCN polynucleotide describedherein. The probe may include a distal tip made from an electricallyconductive material, and the electrode may be coupled to the catheterbody, where the catheter delivers the electrical stimulus to the tissuesite via the electrode coupled to the catheter body and the distal tipof the probe. The catheter may include a pair of electrodes, a firstelectrode coupled to the probe and a second electrode coupled to thecatheter body, where the catheter delivers the electrical stimulus tothe tissue site via the electrode coupled to the catheter body and theelectrode coupled to the probe. The electrical stimulus delivered to thetissue site may include a stimulation pulse or a series of stimulationpulses. The fluid may include a polymer.

As used herein, an “isolated” substance is one that has been removedfrom its natural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. For instance, a polypeptide, apolynucleotide, or a cell can be isolated. Preferably, a substance ispurified, i.e., is at least 60% free, preferably at least 75% free, andmost preferably at least 90% free from other components with which theyare naturally associated.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded RNA and DNA. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidemay be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. A polynucleotide may include nucleotidesequences having different functions, including, for instance, codingregions, and non-coding regions such as regulatory regions.

As used herein, the terms “coding region” and “coding sequence” are usedinterchangeably and refer to a nucleotide sequence that encodes apolypeptide and, when placed under the control of appropriate regulatorysequences, expresses the encoded polypeptide. The boundaries of a codingregion are generally determined by a translation start codon at its 5′end and a translation stop codon at its 3′ end. A “regulatory sequence”is a nucleotide sequence that regulates expression of a coding sequenceto which it is operably linked. Non-limiting examples of regulatorysequences include promoters, enhancers, transcription initiation sites,translation start sites, translation stop sites, and transcriptionterminators. The term “operably linked” refers to a juxtaposition ofcomponents such that they are in a relationship permitting them tofunction in their intended manner. A regulatory sequence is “operablylinked” to a coding region when it is joined in such a way thatexpression of the coding region is achieved under conditions compatiblewith the regulatory sequence.

A polynucleotide that includes a coding region may include heterologousnucleotides that flank one or both sides of the coding region. As usedherein, “heterologous nucleotides” refer to nucleotides that are notnormally present flanking a coding region that is present in a wild-typecell. For instance, a coding region present in a wild-type cell andencoding an HCN polypeptide is flanked by homologous sequences, and anyother nucleotide sequence flanking the coding region is considered to beheterologous. Examples of heterologous nucleotides include, but are notlimited to regulatory sequences. Typically, heterologous nucleotides arepresent in a polynucleotide of the present invention through the use ofstandard genetic and/or recombinant methodologies well known to oneskilled in the art. A polynucleotide of the present invention may beincluded in a suitable vector.

As used herein, an “exogenous polynucleotide” refers to a polynucleotidethat has been introduced into a cell by artificial means. As usedherein, the term “endogenous polynucleotide” refers to a polynucleotidethat is normally or naturally found in a cell. An “endogenouspolynucleotide” is also referred to as a “native polynucleotide.”

The terms “complement” and “complementary” as used herein, refer to theability of two single stranded polynucleotides to base pair with eachother, where an adenine on one strand of a polynucleotide will base pairto a thymine or uracil on a strand of a second polynucleotide, and acytosine on one strand of a polynucleotide will base pair to a guanineon a strand of a second polynucleotide. Two polynucleotides arecomplementary to each other when a nucleotide sequence in onepolynucleotide can base pair with a nucleotide sequence in a secondpolynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. Theterm “substantial complement” and cognates thereof as used herein, referto a polynucleotide that is capable of selectively hybridizing to aspecified polynucleotide under stringent hybridization conditions.Stringent hybridization can take place under a number of pH, salt andtemperature conditions. The pH can vary from 6 to 9, preferably 6.8 to8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium,and other cations can be used as long as the ionic strength isequivalent to that specified for sodium. The temperature of thehybridization reaction can vary from 30° C. to 80° C., preferably from45° C. to 70° C. Additionally, other compounds can be added to ahybridization reaction to promote specific hybridization at lowertemperatures, such as at or approaching room temperature. Among thecompounds contemplated for lowering the temperature requirements isformamide. Thus, a polynucleotide is typically substantiallycomplementary to a second polynucleotide if hybridization occurs betweenthe polynucleotide and the second polynucleotide. As used herein,“specific hybridization” refers to hybridization between twopolynucleotides under stringent hybridization conditions.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers(e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide,enzyme, and protein are all included within the definition ofpolypeptide and these terms are used interchangeably. It should beunderstood that these terms do not connote a specific length of apolymer of amino acids, nor are they intended to imply or distinguishwhether the polypeptide is produced using recombinant techniques,chemical or enzymatic synthesis, or is naturally occurring.

As used herein, “identity” refers to sequence similarity between twopolypeptides or two polynucleotides. The sequence similarity between twopolypeptides is determined by aligning the residues of the twopolypeptides (e.g., a candidate amino acid sequence and a referenceamino acid sequence, such as SEQ ID NO:2, 4, 6, or 8, or a portionthereof) to optimize the number of identical amino acids along thelengths of their sequences; gaps in either or both sequences arepermitted in making the alignment in order to optimize the number ofshared amino acids, although the amino acids in each sequence mustnonetheless remain in their proper order. The sequence similarity istypically at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity. Sequencesimilarity may be determined, for example, using sequence techniquessuch as the BESTFIT algorithm in the GCG package (Madison Wis.), or theBlastp program of the BLAST 2 search algorithm, as described byTatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and availablethrough the World Wide Web, for instance at the internet site maintainedby the National Center for Biotechnology Information, NationalInstitutes of Health. Preferably, sequence similarity between two aminoacid sequences is determined using the Blastp program of the BLAST 2search algorithm. Preferably, the default values for all BLAST 2 searchparameters are used, including matrix=BLOSUM62; open gap penalty=11,extension gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, andoptionally, filter on. In the comparison of two amino acid sequencesusing the BLAST search algorithm, structural similarity is referred toas “identities.”

The sequence similarity between two polynucleotides is determined byaligning the residues of the two polynucleotides (e.g., a candidatenucleotide sequence and a reference nucleotide sequence, such as SEQ IDNO:1, 3, 5, 7, or a portion thereof) to optimize the number of identicalnucleotides along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of shared nucleotides, although the nucleotides in each sequencemust nonetheless remain in their proper order. The sequence similarityis typically at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity. Sequencesimilarity may be determined, for example, using sequence techniquessuch as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector4.5 (Kodak/IBI software package) or other suitable sequencing programsor methods known in the art. Preferably, sequence similarity between twonucleotide sequences is determined using the Blastn program of the BLAST2 search algorithm, as described by Tatusova, et al. (1999, FEMSMicrobiol Lett., 174:247-250), and available through the World Wide Web,for instance at the internet site maintained by the National Center forBiotechnology Information, National Institutes of Health. Preferably,the default values for all BLAST 2 search parameters are used, includingreward for match=1, penalty for mismatch=−2, open gap penalty=5,extension gap penalty=2, gap x_dropoff=50, expect=10, wordsize=11, andoptionally, filter on. In the comparison of two nucleotide sequencesusing the BLAST search algorithm, sequence similarity is referred to as“identities.”

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as an enzymatic reaction, or“suitable” conditions are conditions that do not prevent such eventsfrom occurring. Thus, these conditions permit, enhance, facilitate,and/or are conducive to the event.

As used herein, “genetically modified cell” refers to a cell into whichhas been introduced an exogenous polynucleotide, e.g., an expressionvector. For example, a cell is a genetically modified cell by virtue ofintroduction into a suitable cell of an exogenous polynucleotide that isforeign to the cell, or an exogenous polynucleotide that encodes apolypeptide that is normally present in the cell. “Genetically modifiedcell” also refers to a cell that has been genetically manipulated suchthat endogenous nucleotides have been altered. For example, a cell is agenetically modified cell by virtue of introduction into a suitable cellof an alteration of endogenous nucleotides. For instance, an endogenouscoding region could be mutagenized. Such mutations may result in apolypeptide having a different amino acid sequence than was encoded bythe endogenous polynucleotide. Another example of a genetically modifiedcell is one having an altered regulatory sequence, such as a promoter,to result in increased or decreased expression of an operably linkedendogenous coding region.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Multiple sequence alignment of a human HCN1 (SEQ ID NO:2), ahuman HCN2 (SEQ ID NO:4), a human HCN3 (SEQ ID NO:6), and a human HCN4(SEQ ID NO:8). The six α-helical segments (S1-S6), the pore helix(PoreH), the selectivity filter (SF), and the cyclic-nucleotide bindingdomain (CNBD) are depicted.

FIG. 2. Nucleotide sequence encoding a human HCN1 (SEQ ID NO:1), a humanHCN2 (SEQ ID NO:3), a human HCN3 (SEQ ID NO:5), and a human HCN4 (SEQ IDNO:7), and the corresponding amino acid sequences of the HCN1 (SEQ IDNO:2), the HCN2 (SEQ ID NO:4), the HCN3 (SEQ ID NO:6), and the HCN4 (SEQID NO:8) polypeptides.

FIG. 3. Biological pacemaker from AV node ablated canine injected withthe hHCN4t coding region carried by adenovirus vector. A significantincrease in ventricular heart rate was observed following Adv-hHCN4tadministration. Biological pacemaker reached a peak on day 2 andmaintained a rate at peak level through termination at one week.

FIG. 4. Sequence homology between canine and human HCN4 coding regions.In comparison with each other, human and canine share high similarity inRNA sequence blocks of I, II and III (92, 95%, and 91% respectively).Other areas (in front of I block, between I to II and II to III blocks,and after block III) display significant distinctive sequences withinthese two species.

FIG. 5. Schematic diagram of proposed membrane topology of a HCN channelsubunit.

FIG. 6. Three different super-truncated versions of HCN4 (HCN4st) incomparison with HCN4 wt and HCN4t.

FIG. 7. Schematic diagram of generating super-truncated HCN4 plasmidsfor biological pacemaker gene therapy.

FIG. 8. Confirmation of functionality of HCN4st1 using patch clampexperiments. The panel on the left shows current responses as the cellmembrane is pulsed to increasing hyperpolarizing voltages from apositive holding potential at which HCN4 channels are closed. The rightpanel shows the current-voltage relationship using two different pulseprotocols. The electrophysiology of the channel, including reversalpotential, is unaltered compared to its wild typed (wtHCN4) andtruncated (HCN4t) counterparts.

FIG. 9. Cell image and recordings obtained using a micro-electrode array(MEA) of non-transduced neo-natal rat venetricular myocytes (NRVMs).

FIG. 10. Cell image and MEA recordings obtained from NRVM cellstransduced with 10¹¹ vg/ml of B-AAV1-HCN4st1 virus.

FIG. 11. MEA recordings obtained from NRVMs transduced with 10¹⁰-10⁸vg/ml of B-AAV1-HCN4st1 virus.

FIG. 12. Time course of NRVMs beating rates following transduction withB-AAV1-HCN4st1 at various doses.

FIG. 13. Time course of NRVMs beating rates after transduction withB-AAV1-empty vector at various doses.

FIG. 14. Biological pacemaker from AV node ablated canine injected withhHCN4st1 gene.

FIG. 15. Nucleotide sequence of HCN4 with MyC fusion.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Polypeptides

The present invention includes isolated polypeptides havinghyperpolarization-activated and cyclic nucleotide-gated channelactivity, also referred to herein as channel activity. A polypeptidehaving channel activity is referred to herein as an HCN polypeptide. AnHCN polypeptide includes at least three domains, the transmembrane core,the cytosolic N-terminal domain, and the cytosolic C-terminal domain(Wahl-Schott and Biel, 2009, Coll. Mol. Life Sci., 66:470-494). Thetransmembrane core includes six α-helical segments (S1-S6) and an ionconducting pore loop between S5 and S6 (see FIG. 6). A highly conservedasparagine residue is typically present in the extracellular loopbetween S5 and the pore loop (Much et al., 2003, J. Biol. Chem.278:43781-43786). The pore loop typically includes aglycine-tyrosine-glycine (GYG) motif. The voltage sensor of HCNpolypeptides is formed by a charged S4-helix carrying a series ofarginine or lysine residues regularly spaced at every third position(Vaca et al., 2000, FEBS Lett., 479:35-40), optionally including aserine residue (Chen et al., 2000, J. Biol. Chem., 275:36465-36471).There is evidence suggesting that the loop connecting the S4 with the S5segment plays a role in conferring the differential response to voltage(Long et al., 2005, Science, 309:903-908, Decher et al., 2004, J. Biol.Chem., 279:13859-13865, and Prole et al., 2006, J. Gen. Physiol.128:273-282). The proximal portion of the cytosolic C-terminus mediatesthe sensitivity of HCN polypeptides to cAMP (Zagotta et al., 2003,Nature, 425:200-205). This part of an HCN polypeptide includes a cyclicnucleotide-binding domain of about 120 amino acids (CNBD) and an 80amino acid long linker region that connects the CNBD with the S6segment.

Examples of HCN polypeptides useful in the methods described herein aredepicted at SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8. Insome embodiments an HCN polypeptide useful in the methods disclosedherein is truncated at the amino terminal end. Typically, such atruncation may result in the deletion of amino acids up to, but notincluding, amino acids corresponding to the S1 α-helical segment. Forinstance, with reference to the HCN polypeptide depicted at SEQ ID NO:8,the truncation may be from amino acid 1 to amino acid 258, 257, 256,255, 254, 253, 252, 251, 250, 249, 248, 247, 246, 245, 244, 243, 242,241, 240, 239, 238, 237, 236, 235, 234, 233, 232, 231, 230, 229, 228,227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 217, 216, 215, 214,213, 212, 211, or 210, and so on up to the second amino acid of SEQ IDNO:8. In some embodiments an HCN polypeptide is truncated at the carboxyterminal end. Typically, such a truncation may result in the deletion ofamino acids from the carboxy terminal end up to the amino acids thatmake up the CNBD domain. For instance, with reference to the HCNpolypeptide depicted at SEQ ID NO:8, the truncation may begin at aminoacid 723 (i.e., amino acids 723 to the last amino acid, 1203, are notpresent), 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735,736, 737, 738, 739, or 741, and so on up to amino acid 1202 of SEQ IDNO:8.

In some embodiments, an HCN polypeptide useful in the methods describedherein includes a truncation at both the amino terminal and carboxyterminal ends. Accordingly, an HCN polypeptide of the present inventionmay include an amino acid sequence corresponding to a core regionselected from the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, or SEQ ID NO:8. The core region may be, for instance, aminoacids 139-603 of SEQ ID NO:2, 208-672 of SEQ ID NO:4, 80-545 of SEQ IDNO:6, or 214-723 of SEQ ID NO:8, and may optionally include additionalamino acids located on the amino terminal end and/or the carboxyterminal end. Such additional amino acids may be selected from thecorresponding regions of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQID NO:8, or may be other amino acid sequences as discussed herein. Forinstance, in one aspect an HCN polypeptide may have an amino acidsequence beginning with an amino acid selected from amino acids 92-214of SEQ ID NO:8 and ending with an amino acid selected from amino acids723-1188 of SEQ ID NO:8. Specific examples of HCN polypeptides include,but are not limited to, a core region from SEQ ID NO:8 beginning atamino acid 213, 214, 221, or 226, and ending at amino acid 723, 738, or753.

Other examples of HCN polypeptides include those having sequencesimilarity with a region selected from the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. An HCN polypeptidehaving sequence similarity with the region selected from the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 haschannel activity. An HCN polypeptide may be isolated from a eukaryoticcell, such as a vertebrate cell or an invertebrate cell. Examples ofsuitable vertebrate cells include, but are not limited to, cellsobtained from members of the Family Canidae (such as dogs), members ofthe Family Suidae (such as pigs), members of the Family Muridae (such asrats and mice), members of the genus Ovis (such as sheep), non-humanprimates, and human cells. An HCN polypeptide may be produced usingrecombinant techniques, or chemically or enzymatically synthesized usingroutine methods.

HCN polypeptides form channels that are unique among vertebratevoltage-gated ion channels: they have a reverse voltage dependence thatleads to activation upon hyperpolarization. In addition,voltage-dependent opening of these channels is directly regulated by thebinding of cAMP. HCN polypeptides activate upon hyperpolarization with acharacteristic sigmoidal time course. An HCN polypeptide having channelactivity will display a reverse voltage dependence that leads toactivation upon hyperpolarization under suitable conditions. Suitableconditions typically include the expression of an HCN polypeptide in acell such that it can form channels, and then assaying for activationupon hyperpolarization. Suitable assays include whole cell patch clampanalysis and microelectrode array. Preferably, whole cell patch clampanalysis is used. Methods for whole cell patch clamp analysis are knownand routine in the art (Hamill, 1981, Pflugers Arch., 391(2):85-100). Anexample of a whole cell patch clamp assay is described in Example 1. Apolypeptide that leads to functional HCN4 current under suitableconditions is considered to have channel activity and to be an HCNpolypeptide, while a polypeptide that does not lead to functional HCN4current under suitable conditions is considered to not have channelactivity and is not an HCN polypeptide.

The amino acid sequence of an HCN polypeptide having sequence similarityto a region of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8 mayinclude conservative substitutions. A conservative substitution istypically the substitution of one amino acid for another that is amember of the same class. For example, it is well known in the art ofprotein biochemistry that an amino acid belonging to a grouping of aminoacids having a particular size or characteristic (such as charge,hydrophobicity, and/or hydrophilicity) may generally be substituted foranother amino acid without substantially altering the secondary and/ortertiary structure of a polypeptide. For the purposes of this invention,conservative amino acid substitutions are defined to result fromexchange of amino acids residues from within one of the followingclasses of residues: Class I: Gly, Ala, Val, Leu, and Ile (representingaliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile, Ser, and Thr(representing aliphatic and aliphatic hydroxyl side chains); Class III:Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys andMet (representing sulfur-containing side chains); Class V: Glu, Asp, Asnand Gln (carboxyl or amide group containing side chains); Class VI: His,Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro,Trp, Tyr, Ile, Val, Leu, Phe and Met (representing hydrophobic sidechains); Class VIII: Phe, Trp, and Tyr (representing aromatic sidechains); and Class IX: Asn and Gln (representing amide side chains). Theclasses are not limited to naturally occurring amino acids, but alsoinclude artificial amino acids, such as beta or gamma amino acids andthose containing non-natural side chains, and/or other similar monomerssuch as hydroxyacids. SEQ ID NOs:2, 4, 6, and 8 are shown in FIG. 1 in amultiple protein alignment. Identical and conserved amino acids aremarked. Also depicted are those regions corresponding to the sixα-helical segments, the PoreH, the SF, and the CNBD domains. Thisinformation, and other information regarding conserved regions of HCNpolypeptides described herein, permit the skilled person to predictwhether alterations to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8 are likely to result in an HCNpolypeptide having channel activity.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of apolypeptide sequence to change. The first method relies on the processof evolution, in which mutations are either accepted or rejected bynatural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselects or screens to identify sequences that maintain functionality. Asstated by the authors, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which changes are likely to be permissive at a certain positionof the protein. For example, most buried amino acid residues requirenon-polar side chains, whereas few features of surface side chains aregenerally conserved. Other such phenotypically silent substitutions aredescribed in Bowie et al, and the references cited therein.

In some aspects an HCN polypeptide may include specific mutations in theregion linking the S3 and S4 segments (e.g., amino acids 360-367 of SEQID NO:8). Such mutations are depicted in Table 1 (see Sigg et al., U.S.Patent Application Publication 2009/0099611.

TABLE 1 S3-S4 linker SEQ ID (amino acids NO: Mutant 360-368)  9Wild-type HCN4 ETRIDSEVY 10 T360A EARIDSEVY 11 Δ363-367 ETRI 12T360A, Δ363-367 EARI 13 TRI360-362AGM EAGMDSEVY 14 TRI360-362KGMEKGMDSEVY 15 T360A, I362M EARMDSEVY 16 T360A, Δ365-367 EARIDS 17 E365GETRIDSGVY 18 E365A ETRIDSAVY 19 R361G ETGIDSEVY 20 TR360-361AA EAAIDSEVY21 I362C ETRCDSEVY 22 I362S ETRSDSEVY 23 I362T ETRTDSEVY 24TRI360-362AGM, EAGM Δ363-367

An HCN polypeptide may be expressed as a fusion polypeptide thatincludes an HCN polypeptide and an additional amino acid sequence. Forinstance, the additional amino acid sequence may be useful forpurification of the fusion polypeptide by affinity chromatography.Various methods are available for the addition of such affinitypurification moieties to proteins. Examples of affinity tags include apolyhistidine-tag and a MyC-tag (see, for instance, Hopp et al. (U.S.Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No. 4,782,137), Sgarlato(U.S. Pat. No. 5,935,824), and Sharma Sgarlato (U.S. Pat. No.5,594,115)). In another example, the additional amino acid sequence maybe useful fortagging the fusion polypeptide to aid in identification ofthe polypeptide in various conditions, including in a tissue or in acell.

Polynucleotides

The present invention also includes isolated polynucleotides encoding anHCN polypeptide described herein. A polynucleotide encoding apolypeptide having channel activity is referred to herein as an HCNpolynucleotide. HCN polynucleotides may have a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or region of one ofthose amino acid sequences, as described herein. An example of the classof nucleotide sequences encoding a region of a polypeptide disclosed atSEQ ID NO:2, 4, 6, or 8 is the corresponding region of SEQ ID NO:1, 3,5, or 7, respectively. It should be understood that a polynucleotideencoding an HCN polypeptide represented by, for instance, SEQ ID NO:8,or a portion thereof, is not limited to the nucleotide sequencedisclosed at SEQ ID NO:7, but also includes the class of polynucleotidesencoding such polypeptides as a result of the degeneracy of the geneticcode. For example, the naturally occurring nucleotide sequence SEQ IDNO:7 is but one member of the class of nucleotide sequences encoding apolypeptide having the amino acid sequence SEQ ID NO:8. The class ofnucleotide sequences encoding a selected polypeptide sequence is largebut finite, and the nucleotide sequence of each member of the class maybe readily determined by one skilled in the art by reference to thestandard genetic code, wherein different nucleotide triplets (codons)are known to encode the same amino acid. It should be understood thatany nucleotide sequence taught herein also includes the complementthereof, and the corresponding RNA sequences.

An HCN polynucleotide of the present invention may have sequencesimilarity with the nucleotide sequence of SEQ ID NO:1, 3, 5, or 7. HCNpolynucleotides having sequence similarity with the nucleotide sequenceof SEQ ID NO:3 encode an HCN polypeptide. An HCN polynucleotide may beisolated from a vertebrate cell or an invertebrate cell, or may beproduced using recombinant techniques, or chemically or enzymaticallysynthesized. An HCN polynucleotide may further include heterologousnucleotides flanking the open reading frame encoding the HCNpolypeptide. Typically, heterologous nucleotides may be at the 5′ end ofthe coding region, at the 3′ end of the coding region, or thecombination thereof. The number of heterologous nucleotides may be, forinstance, at least 10, at least 100, or at least 1000.

HCN polynucleotides may be obtained from a eukaryotic cell, such as avertebrate cell or an invertebrate cell. Examples of suitable vertebratecells include, but are not limited to, cells obtained from members ofthe Family Canidae, and human cells. HCN polynucleotides may be producedin vitro or in vivo. For instance, methods for in vitro synthesisinclude, but are not limited to, chemical synthesis with a conventionalDNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotidesand reagents for such synthesis are well known.

An HCN polynucleotide may be present in a vector. A vector is areplicating polynucleotide, such as a plasmid, phage, or cosmid, towhich another polynucleotide may be attached so as to bring about thereplication of the attached polynucleotide. Construction of vectorscontaining a polynucleotide of the invention employs standard ligationtechniques known in the art. See, e.g., Sambrook et al, MolecularCloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press(1989). A vector may provide for further cloning (amplification of thepolynucleotide), i.e., a cloning vector, or for expression of thepolynucleotide, i.e., an expression vector. The term vector includes,but is not limited to, plasmid vectors, viral vectors, cosmid vectors,and artificial chromosome vectors. In some aspects preferred vectorsinclude those useful for gene therapy, e.g., vectors that can beadministered to a subject to result in transient or sustained expressionof a coding region to result in a beneficial polypeptide. A largevariety of such vectors are known in the art and are readily available.Examples of vectors useful in gene therapy include isolated nucleicacid, e.g., plasmid-based vectors which may be extrachromosomallymaintained, including minicircle vectors; viral vectors, e.g.,recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus,papilloma virus, or adeno-associated virus; and transposons, e.g.,recombinant transposons such as Sleeping Beauty and piggyBac. Preferredexamples of vectors include single strand adeno-associated virus (ssAAV)and self complementary AAV (scAAV), a modified adeno-associated virusthat bypasses the required second-strand DNA synthesis to achievetranscription of the coding region (McCarty, 2008, Mol Ther.,16:1648-1656). Useful ssAAV vectors and scAAV vectors are commerciallyavailable from, for instance, Virovek, Hayward, Calif.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. In some aspects, suitable host cells forcloning or expressing the vectors herein include eukaryotic cells.Suitable eukaryotic cells include fungi and mammalian cells. In otheraspects, suitable host cells for cloning or expressing the vectorsherein include prokaryotic cells. Suitable prokaryotic cells includeeubacteria, such as gram-negative microbes, for example, E. coil.Vectors may be introduced into a host cell using methods that are knownand used routinely by the skilled person. For example, calcium phosphateprecipitation, electroporation, heat shock, lipofection, microinjection,and viral-mediated nucleic acid transfer are common methods forintroducing nucleic acids into host cells.

An expression vector optionally includes regulatory sequences operablylinked to the coding region. An example of a regulatory sequenceincludes a promoter. The invention is not limited by the use of anyparticular promoter, and a wide variety of promoters are known.Promoters act as regulatory signals that bind RNA polymerase in a cellto initiate transcription of a downstream (3′ direction) coding region.The promoter used may be a constitutive or an inducible promoter. It maybe, but need not be, heterologous with respect to the host cell.

In some aspects, tissue-specific promoters may be used. Tissue-specificexpression may enhance the safety of a therapy described herein asexpression in non-target tissue becomes less likely. For example,cardiac tissue specific promoters allow cardiac myocyte specificexpression of the coding region of interest (including expression instem cells with cardiac phenotype). Examples of cardiac tissue specificpromoters include, but are not limited to, promoters from the followingcoding regions: an α-myosin heavy chain coding region, e.g., aventricular α-myosin heavy chain coding region, β-myosin heavy chaincoding region, e.g., a ventricular β-myosin heavy chain coding region,myosin light chain 2 v coding region, e.g., a ventricular myosin lightchain 2 coding region, myosin light chain 2 a coding region, e.g., aventricular myosin light chain 2 coding region, cardiomyocyte-restrictedcardiac ankyrin repeat protein (CARP) coding region, cardiac α-actincoding region, cardiac m2 muscarinic acetylcholine coding region, ANPcoding region, BNP coding region, cardiac troponin C coding region,cardiac troponin I coding region, cardiac troponin T coding region,cardiac sarcoplasmic reticulum Ca-ATPase coding region, and skeletalα-actin coding region. Further, chamber-specific promoters or enhancersmay also be employed, e.g., for atrial-specific expression, the quailslow myosin chain type 3 (MyHC3) or ANP promoter may be used. Examplesof ventricular myocyte-specific promoters include a ventricular myosinlight chain 2 promoter and a ventricular myosin heavy chain promoter.

Other useful promoters, for example, would be sensitive to electricalstimulus that could be provided from, for example, an implantabledevice. Electrical stimulation can promote gene expression (Padua etal., U.S. Patent Application No. 2003/0204206 A1).

Other regulatory regions include drug-sensitive elements (e.g., adrug-inducible suppressor or promoter). Drug-responsive promoters mayinduce or suppress expression of an operably linked coding region. Forexample, a tetracycline responsive element (TRE) that binds doxycyclinemay present within a promoter construct. When doxycycline is removed,transcription from the TRE is turned off in a dose-dependent manner.Examples of inducible drug-responsive promoters are theecdysone-inducible promoter (Johns and Marban, U.S. Pat. No. 6,214,620)and rapamycin-dependent expression (Clackson et al., U.S. Pat. No.6,506,379, see also Discher et al., 1998, J. Biol. Chem.,273:26087-26093; Prentice et al., 1997, Cardiovascular Res., 35:567-576).

Further examples of regulatory regions include enhancers, such ascardiac enhancers, to increase the expression of an operably linkedcoding region in cardiac tissue, such as regions of the cardiacconduction system. Such enhancer elements may include the cardiacspecific enhancer elements derived from Csx/Nk×2.5 regulatory regions(Lee and Izumo, U.S. Patent Application 2002/0022259) or the cGATA-6enhancer.

An expression vector may optionally include a ribosome binding site anda start site (e.g., the codon ATG) to initiate translation of thetranscribed message to produce the polypeptide. It may also include atermination sequence to end translation. A termination sequence istypically a codon for which there exists no correspondingaminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotideused to transform the host cell may optionally further include atranscription termination sequence.

A vector introduced into a host cell optionally includes one or moremarker sequences, which typically encode a molecule that inactivates orotherwise detects or is detected by a compound in the growth medium. Forexample, the inclusion of a marker sequence may render the transformedcell resistant to an antibiotic, or it may confer compound-specificmetabolism on the transformed cell. Selectable markers can be positive,negative or bifunctional. Positive selectable markers allow selectionfor cells carrying the marker, whereas negative selectable markers allowcells carrying the marker to be selectively eliminated. A variety ofsuch marker genes have been described, including bifunctional (i.e.,positive/negative) markers (see, e.g., Lupton, PCT Publication Nos. WO92/08796 and WO 94/28143). Such marker genes can provide an addedmeasure of control that can be advantageous in gene therapy contexts.

Polypeptides useful in the present invention may be produced usingrecombinant DNA techniques, such as an expression vector present in acell. Such methods are routine and known in the art. The polypeptidesmay also be synthesized in vitro, e.g., by solid phase peptide syntheticmethods. The solid phase peptide synthetic methods are routine and knownin the art. A polypeptide produced using recombinant techniques or bysolid phase peptide synthetic methods may be further purified by routinemethods, such as fractionation on immunoaffinity or ion-exchangecolumns, ethanol precipitation, reverse phase HPLC, chromatography onsilica or on an anion-exchange resin such as DEAE, chromatofocusing,SDS-PAGE, ammonium sulfate precipitation, gel filtration using, forexample, Sephadex G-75, or ligand affinity.

The present invention also includes genetically modified cells that havean HCN polynucleotide encoding an HCN polypeptide. Compared to a controlcell that is not genetically modified according to the presentinvention, a genetically modified cell may exhibit production of an HCNpolypeptide. A polynucleotide encoding an HCN polypeptide may be presentin the cell as an extrachromosomal vector or integrated into achromosome. Examples of cells include, but are not limited to, excitablecells, such as cardiomyocytes and HL-5 cells; and non-excitable cells,such as stem cells, fibroblasts, mesenchymal cells, and HEK293 cells. Insome aspects the cells may also be modified to express connexions or gapjunctions. The coding regions encoding connexion polypeptides arereadily available to the skilled person.

A genetically modified cell may be ex vivo or in vivo. “Ex vivo” refersto a cell that has been removed from the body of an animal. Ex vivocells include, for instance, primary cells (e.g., cells that haverecently been removed from a animal and are capable of limited growth intissue culture medium), and cultured cells (e.g., cells that are capableof long term culture in tissue culture medium). “In vivo” refers tocells that are present within the body of an animal.

Compositions

The present invention is also directed to compositions including an HCNpolynucleotide, HCN polypeptide, or genetically modified cell. Suchcompositions typically include a pharmaceutically acceptable carrier. Asused herein “pharmaceutically acceptable carrier” includes saline,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Additional activecompounds can also be incorporated into the compositions.

A composition may be prepared by methods well known in the art ofpharmacy. In general, a composition can be formulated to be compatiblewith its intended route of administration. Solutions or suspensions caninclude the following components: a sterile diluent such as water foradministration, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfate; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; electrolytes, such as sodium ion, chloride ion, potassiumion, calcium ion, and magnesium ion, and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Acomposition can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Compositions can include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile solutionsor dispersions. For intravenous administration, suitable carriersinclude physiological saline, bacteriostatic water, Cremophor EL™ (BASF,Parsippany, N.J.) or phosphate buffered saline. A composition istypically sterile and, when suitable for injectable use, should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. Prevention of the action of microorganisms can be achieved byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compound(e.g., an HCN polynucleotide, or, in some aspects, an HCN polypeptide orgenetically modified cell) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehicle,which contains a dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

An active compound may be administered by any route including, but notlimited to, intramuscular, buccal, rectal, intravenous or intracoronaryadministration, and transfer to cells may be enhanced usingelectroporation and/or iontophoresis. Administration may be systemic orlocal. In some aspects local administration may have advantages forsite-specific, targeted disease management. Local therapies may providehigh, clinically effective concentrations directly to the treatmentsite, without causing systemic side effects. Examples of locations towhich an active compound can be targeted include, but are not limitedto, the right or left atrium, including the sinoatrial node, the rightor left ventricle, including the atrioventricular node. For instance,endocardial or myocardial cells of an atrium or a ventricle may betargeted. In some aspects, an HCN polynucleotide can be implanted in, ordownstream from, the conduction pathway, in a heart region that isexperiencing or may experience poor conduction. For example, if cardiaccontraction is not being properly initiated by the SA node but the AVnode conduction is intact, an HCN polynucleotide may be implanted in themyocardium of the SA node or the right atrium to cause the targetedregion to depolarize and create electric impulses that will travel tothe AV node. Alternatively, if cardiac contraction is not being properlyconducted by the AV node then an HCN polynucleotide may be implanteddownstream in the conduction pathway from the right atrium, i.e. in thebundle of His, the Purkinje network, or one of the ventricles. Otherdelivery sites include, but are not limited to, left ventricularepicardium.

Examples of routes of administration include the use of a delivery tool,such as a syringe for direct injection into cardiac tissue (forinstance, during open heart surgery) or by catheter. For instance, onetype of catheter useful in the methods described herein has electricsensing capabilities, which permits introduction of an active compounddirectly into the targeted cardiac tissue. The delivery tool may includeelectrodes for sensing electric activity and delivering pacing stimuliin order to determine the desired location for the biologicalpacemakers. Once the location is determined, an active compound isdelivered to the cardiac tissue. The delivery tool may include aninjection device that injects the active compound into cardiac tissue.One suitable method for injecting a genetic construct directly into themyocardium is described by Guzman et al., 1993, Circ. Res.,73:1202-1207. Furthermore, a delivery system for delivering geneticmaterial to a targeted heart region is described in Laske et al. (U.S.Pat. No. 7,103,418) and Stokes et al. (PCT Publication No. WO 98/02150).Systems for myocardial, endocardial, sub-epicardial and epicardialdelivery are described in Sullivan and Hezi-Yamit (U.S. Published PatentApplication 20100137976), Hiniduma-Lokuge et al. (PCT Publication No.WO/2008/055001), and Sommer et al., (U.S. Pat. No. 7,274,966 and U.S.Pat. No. 7,187,971). Alternatively, genetically engineered cells may becultured and proliferated on a solid scaffold, and then surgicallydelivered to the selected heart region together with the scaffold. Thescaffold may also be directly injected into cardiac tissue.

Perfusion protocols that are useful are often sufficiently capable ofdelivering a genetic construct to at least about 10% of cardiacmyocytes. Infusion volumes of between 0.01 ml and 3 ml are useful fordirect intramyocardial injection. Also, suitable methods for targetingnon-viral vector genetic constructs to the heart are described inLawrence (U.S. Pat. No. 6,376,471).

When a polynucleotide is introduced into cardiac cells using anysuitable technique, the polynucleotide is delivered into the cells by,for example, transfection or transduction procedures. Transfection andtransduction refer to the acquisition by a cell of new genetic materialby incorporation of added polynucleotides. Transfection can occur byphysical or chemical methods. Many transfection techniques are known tothose of ordinary skill in the art including, without limitation,calcium phosphate DNA co-precipitation, DEAE-dextrin DNA transfection,electroporation, naked plasmid adsorption, and cationicliposome-mediated transfection (commonly known as lipofection).Transduction refers to the process of transferring nucleic acid into acell using a DNA or RNA virus.

A polynucleotide described herein may be used in combination with otheragents assisting the cellular uptake of polynucleotides, or assistingthe release of poylnucleotides from endosomes or intracellularcompartments into the cytoplasm or cell nuclei by, for instance,conjugation of those to the polynucleotide. The agents may be, but arenot limited to, peptides, especially cell-penetrating peptides, proteintransduction domains, and/or dsRNA-binding domains which enhance thecellular uptake of polynucleotides (Dowdy et al., US Published PatentApplication 2009/0093026, Eguchi et al., 2009, Nature Biotechnology27:567-571, Lindsay et al., 2002, Curr. Opin. Pharmacol., 2:587-594,Wadia and Dowdy, 2002, Curr. Opin. Biotechnol. 13:52-56. Gait, 2003,Cell. Mol. Life Sci., 60:1-10). The conjugations can be performed at aninternal position at the oligonucleotide or at a terminal postionseither the 5′-end or the 3′-end.

A polynucleotide described herein may be present in liposomes, includingneutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE orDMRIE/DOPE liposomes, and/or associated with other molecules such asDNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes orpolyethyleneimine (PEI). An active compound may be present in a polymermatrix, for instance, a polymer matrix may be formed of anyphysiologically compatible material which generally retains apolynucleotide (which is a charged molecule) or optionally other agentsincluding other agents under physiological conditions for a sustainedperiod of time. The polymer matrix may extrude (release) thepolynucleotide in response to an external stimulus, such as an electricfield created by an electrical signal, or the matrix may provide forpassive delivery.

Toxicity and therapeutic efficacy of such active compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the ED₅₀ (the dosetherapeutically effective in 50% of the population). The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for use in humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For a compound used in the methods of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. In those aspects where a viral vector is used, such asan AAV-based vector, a dosage may be at least 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³, or 10¹⁴ viral particles.

Administration of an HCN polynucleotide, an HCN polypeptide, or agenetically modified cell described herein may be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to a person skilled in the art.The administration may be essentially continuous over a preselectedperiod of time or may be in a series of spaced doses.

Methods of Use

The present invention also includes methods of using the HCNpolynucleotides, HCN polypeptides, and genetically modified cellsdescribed herein. The methods include, for instance, methods of treatinga cardiac pacing condition. Examples of cardiac pacing conditionsinclude, but are not limited to, patients with atrioventricular (AV)node dysfunction and/or sinoatrial (SA) node dysfunction. Such patientsmay have bradyarrhythmia, such as those with the clinical syndrome sicksinus syndrome. Cardiac pacing conditions may also occur in patientsundergoing surgery (such as coronary artery bypass surgery or insertionof an artificial heart valve), or having endocarditis. Signs andsymptoms associated with cardiac pacing conditions and the evaluation ofsuch signs and symptoms are routine and known in the art. As usedherein, the term “symptom” refers to subjective evidence of a cardiacpacing condition experienced by a subject. As used herein, the term“clinical sign” or, simply, “sign” refers to objective evidence of acardiac pacing condition.

Treatment may be prophylactic or, alternatively, may be initiated aftera cardiac pacing condition is evident. Treatment that is prophylactic,for instance, initiated before an animal manifests symptoms of a cardiacpacing condition, is referred to herein as treatment of a patient thatis “at risk” of developing a cardiac pacing condition. Treatmentinitiated after development of symptoms of a cardiac pacing conditionmay result in decreasing the severity of the symptoms, or completelyremoving the symptoms. An “effective amount” is an amount effective toprevent the manifestation of symptoms of a cardiac pacing condition,decrease the severity of the symptoms of cardiac pacing condition,and/or completely remove the symptoms. It is not required that anycomposition of the present invention completely remove or cure allsymptoms of a cardiac pacing condition.

Treatment may result in increasing the intrinsic pacing rate of cardiaccells, preferably to the normal physiological range for the subject. Insome aspects, the intrinsic pacing rate is increased to 60-80 beats perminute (bpm) at rest and 90-115 bpm during moderate exercise.

The methods may include administering a composition of the presentinvention to a subject in need thereof. The subject may be human, or ananimal, such as an animal typically used as a model in the study andevaluation of treatments for a cardiac pacing condition, such as a dogor pig, sheep, or non-human primate.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example 1

This example describes the development a super-truncated HCN4 constructin order to be packaged in size limited scAAV vector and to beapplicable across species and between human without inducing an immuneresponse. Further, an AAV1 vector carrying this super-truncated HCN4 wasto be generated by exploring an alternative AAV generation technology.The pace maker function was confirmed by ex vivo evaluation.

BACKGROUND

Previously we had demonstrated biological pacemaker function using bothhuman full length HCN4 polypeptide (wtHCN4) and a human C-terminaltruncated HCN4 version (HCN4t) having amino acids 1-738 of SEQ ID NO:8,packaged in an adenovirus vector (Adv). We used Adv as a carrier todeliver human hyperpolarization-activated cyclic-nucleotide-gated (HCN)ion channel 4 and established proof of concept that HCN4t, along withthe full length HCN4, are capable of generating a ventricular biologicalpacemaker in an AV blocked canine model (FIG. 3).

Although Adv has shown remarkable efficacy in gene transfer, wedeveloped a clinically relevant vector, traditional adeno-associatedvirus vector (AAV), to carry a biological pacemaker coding region, HCN4.In comparison with Adv, traditional AAV, also referred to assingle-stranded AAV (ssAAV), is a very attractive vector for cardiacgene therapy because of lack of pathogenicity, very low immunogenicity,and long-term gene transfer potential with decreased risk of malignanttransformation in small and large animal models.

However, the problems associated with ssAAV vector include a latentperiod from 10 days to 1 month before ssAAV mediated gene expressionpeaks. While a 3-4 week waiting period required for ssAAV to reach thesteady state may be acceptable for one-time life-saving therapy such asa biological pacemaker, a faster expressing system would certainly bemost applicable clinically since it would minimize the time a patient ison electronic pacing as a bridge, and would also provide much quickerfeedback to the physician if the biological pacemaker were to need anyintervention (e.g., to adjust pacing rate). Therefore, it is desirableto determine whether a different type of AAV vector system, theself-complementary AAV vector system (scAAV) will function.

The traditional AAV system contains single stranded DNA and requireshost cell synthesis of the complementary strand. Therefore there is alag period, when the expression is generally quite slow with the peakfunction occurring 3-4 weeks after the gene administration. In contrast,scAAV system contains double strand DNA and therefore eliminates therate-limiting step of second-strand DNA synthesis. Thus, scAAV canattain peak expression in a matter of days. This fast ramp-up featuremakes scAAV a very promising vector for clinical applications.

ssAAV can package a coding region and regulatory sequences (includingtwo ITR regions, promoter, transgene, and poly-A signal) roughly thesize of 4.8 kilo-base pairs (kb) and allows packaging of HCN4t (2,214base pairs (bp) for HCN4t versus 3,612 by for wtHCN4). As for scAAV, itcan only package ˜2.5 kb of coding region and regulatory sequences.While much shorter than in its wild-type counterpart, the coding regionencoding HCN4t is still longer than the packaging capacity of scAAV. Wedeveloped an even shorter version of HCN4 construct, so that thepackaging in scAAV vector system is feasible. The shorter version wasdesignated super truncated HCN4 (HCN4st).

In addition to the limitation related to their large sizes, anotherlimitation of the wtHCN4 and HCN4t genes is that they have regions thatshow inter-species variability. For example, between canine and humangenes, there is a considerable lack of homology in the 5′ region of theHCN4 gene (FIG. 4). The HCN4st removes all the regions that exhibitinter-species variability and this would imply that the risk ofgenerating an immune response and loss of pacemaker function would beminimized with an HCN4st gene. There are greater than 98% identicalresidues between human and canine HCN4st1 sequence.

HCN4 channels (FIG. 5) consist of 6 transmembrane domains, with a poreregion between S5 and S6 and a cyclic nucleotide-binding domain (CNBD)in the cytoplasmic C-terminal region. While HCN4t deleted nucleotidesafter the CNBD region, HCN4st was designed to delete regions from theamino terminal end, but not extend into the S1 region. It is withinblock III of FIG. 4.

In an effort to generate scalable production of AAV vectors, as well asto overcome the toxicity of HCN4 in routine host cells, such as HEK293,a “BAC-to-AAV” technology (Chen U.S. Published Patent Application20090203071) that uses a baculovirus expression system to produce AAVvectors in insect cells under serum-free condition was used. BAC-to-AAVtechnology enhances the packaging of much more VP1 proteins into thevirions than other systems and therefore greatly increases theperformance of AAV vectors.

Methods:

Generating Super-Truncated Version of HCN4 (HCN4st).

HCN4t construct was generated by truncating 1,398 base pairs (bp)nucleotides that encode the C-terminus of wild type HCN4. The truncationsite was chosen such that a cyclic nucleotide-binding domain (CNBD) waspreserved in the C-terminus region of HCN4t. HCN4st constructs includeda series of deletions on the N-terminus of the HCN4t, in front of thesix transmembrane domains (FIG. 6).

Each individual HCN4st gene was generated via PCR amplification by usingHCN4t as template. Sequence specific primers were designed using thenucleotide sequence available at Genbank accession NM_(—)005477.2.Upstream primers HCN4st were 5′ ata gcg cga att ccc gcc atg cag cgc cagttc ggg (SEQ ID NO:25), 5′ ata gcg cga att ccc gcc atg ctc caa ccc ggg g(SEQ ID NO:26) and 5′ ata gcg cga att ccc gcc atg ttc ggc agc cag aaa g(SEQ ID NO:27) respectively for HCN4st1, st2 and st3. The codingsequence encoding HCN4st1 encodes a polypeptide corresponding to aminoacids 214-719 of SEQ ID NO:8 (FIG. 1), the coding sequence encodingHCN4st2 encodes a polypeptide corresponding to amino acids 221-719 ofSEQ ID NO:8, and the coding sequence encoding HCN4st3 encodes apolypeptide corresponding to amino acids 233-719 of SEQ ID NO:8. Thedownstream primer for each HCN4st was 5′ cggcggatcccctagagatat (SEQ IDNO:28). MyC sequence (AGCAGAAGCTGATCTCAGAGGAGGACCTGCTT, SEQ ID NO:29)was attached at the C-terminal of different HCN4t modifications. ThreeHCN4st genes were cloned into a pTopo sequence plasmid (Invitrogen,Carlsbad, Calif., USA) and verified by restriction enzyme analysis (NewEngland Biolabs, Ipswich, Mass., USA) and the nucleotide sequence wasconfirmed (Eurofins MWG Operon, Huntsville, Ala. USA). HCN4st genes weresubsequently subcloned into the expression plasmid pIRES-EGFP(Clonetech, Mountain View, Calif., USA) for functional testing by patchclamp. Selected HCN4st genes were further subcloned into an AAV shuttleplasmid (ViroVek, Hayward, Calif., USA) for making B-AAV-HCN4st1 vector(FIG. 7, and described below).

Patch Clamp

HEK 293 Cell Transfection

Human embryonic kidney (HEK293, ATCC) cells were split on 12 well plateone day before transfection. Three pHCN4st-IRES-EGFP plasmids weretransfected into 70% confluent HEK293 cells with Fugene 6 (Roche)transfection reagents, along with pHCN4t-IRES-EGFP. The transfectedcells were replated on gelatin-fibronectin coated glass coverslips 24hours after transfection.

Whole Cell Patch Clamp

One day after reculture, the cells plated on a coverslip weretransported to a chamber mounted on the stage of a Nikon microscope. Thechamber was continuously superfused (˜0.5 ml/min) with the Tyrodesolution. The whole-cell configuration of the patch-clamp technique(Hamill et al., 1981, Pflugers Arch. 391(2):85-100) was applied.Briefly, glass electrodes (World Precision Instruments, Sarasota, Fla.,USA) with 3 to 5 MΩ resistance were connected via an Ag-AgCl wire to anAxopatch 200A amplifier interfaced with a DigiData-1322 acquisitionsystem. After forming a conventional “gigaohm” seal, electrodecapacitance was compensated. Additional suction ruptured the patchedmembrane and formed the whole-cell configuration. Cell membranecapacitance (C_(m)) was measured in each patched cells with the pCLAMPprogram (version 9.2, Axon Instruments, Foster City, Calif., USA).

The hyperpolarization-activated cyclic nucleotide-gated inward current(I_(h)) was measured with the modified Tyrode bath solution. I_(h) wasevoked by 5 s hyperpolarizing steps to potentials ranging from 0 to −140mV from a holding potential of −40 mV. The reversal potential of I_(h)was evaluated by tail currents recorded by 3 s ‘tail’ steps to membranepotentials ranging from −80 to 20 mV in 10 mV increments followed by a 5s conditioning potential step to −130 mV every 15 s. The holdingpotential was set at −40 mV. The activation of I_(h) was elicited by 3 s‘tail’ pulses to −130 mV followed 5 s conditioning pulses from 0 mV to−140 mV in 10 mV increments. The pulse rate was every 30 s.

Data Analysis

Data were collected with the pCLAMP software (version 9.02). I_(h) wasevaluated at a point near the end of each test pulse unless statedotherwise (tail-current measurements). The current amplitudes werenormalized with respect to the corresponding values of C_(m) to minimizethe current difference due to cell size. A single-exponential fit(Axon-Clampfit 9.02) of current traces allowed derivation of timeconstants (τ) of current activation and deactivation. Some data werefitted by a Boltzmann equation {1/[1+exp(V_(1/2)−V)/k], where V_(1/2) isthe half-inactivation potential, V is the voltage potential, and k isthe slope factor (in mV/e-fold change in current)}. The best-fitprocedure was performed with a commercial software program (Origin 7.0,Microcal™ Software Inc., Northampton, Ma., USA). All data are presentedas mean±standard error of the mean unless otherwise stated. UnpairedStudent's t-test was applied for statistical analysis as appropriate.Differences were considered significant if P≦0.05.

B-AAV1-HCN4st1 Generation

Following patch clamp, functional HCN4st gene was further subcloned andthe polynucleotide sequence depicted in FIG. 15 was used as a templatefor PCR using the primers 5′ ata gcg cga att ccc gcc atg cag cgc cag ttcggg (SEQ ID NO:25) and 5′ cggcggatcccctagagatat (SEQ ID NO:28), and theamplification product was used by ViroVek to produce an AAV1 vector thatencoded an HCN4st1 polypeptide that included a MyC sequence fused to theamino terminal end of the polypeptide. The methods used by ViroVekinclude cloning gene of interest into an pFB-AAV shuttle plasmid,generation of Bacmid and purification of Bacmid DNA, transfection of Sf9cells to generate baculovirus, amplification of baculovirus andtitration, production of AAV and CsCl purification, and desalting,filter sterilization, and AAV titration (see Chen, U.S. Published PatentApplication 20090203071, U.S. Provisional Patent Application 60/839,761,and International Application PCT/US07/76799, andhttp://www.virovek.com/AAV_Production.html. Together with AAV repcomponent and vp component, this shuttle vector were co-transfected intoSF9 insect cells for generation of recombinant baculovirus andproduction of a virus vector containing a coding region encodingHCN4st1, designated B-AAV1-HCN4st1, where the “B” refers to generationusing the baculovirus system. Following purification of the virus vectorthrough buffer exchange and sterile filtration, virus titer wasdetermined by qPCR. The resulting B-AAV1-HCN4st1 was dissolved in PBSwith 0.001% Pluronic F-68 buffer at concentration of 1.03E+13 vg/mL.Endotoxin level was tested by Biotest Labs with <1EU/ml.

Microelectrode Array (MEA) Measurements

Primary Cell Isolation

Neonatal rats were sacrificed by decapitation and hearts were rapidlyexcised and washed in Ca²⁺ free balanced salt solution. The ventricleswere minced into 1-2 mm³ pieces and dissociated into single cellsuspension by repeated digestion with proteolytic enzymes. Eachdigestion, enhanced with gentle shaking, lasted for 15-20 minutes. Thenmyocytes were mechanically dispersed by triturating. The undigestedmasses and first few digestion fractions were discarded after filteringthrough a cell strainer. Collected cells suspension was mixed with serumfor enzyme deactivation, centrifuged, and re-suspended in culturingmedia. Cells were additionally incubated with 10 μg/ml of DNAse for 10min at 37° C. For fibroblasts separation and myocytes enrichment two 1.5hrs pre-plating steps (incubation of cell suspension in 75 cm² flasks)were performed. After pre-plating, slowly attaching myocytes wereseparated from quickly attaching fibroblasts, collected, counted,re-suspended, and plated in pretreated MEAs (at −500000 cells/ml,−180000 cells/cm²). Each MEA had ˜64 electrodes spaced ˜100 μm andcovering a total area of approximately 0.5 mm².

NRVMs were cultured with Norepinephrine and Bromodeoxyuridine (BrDu) for2 days, than with BrDu for 2 more days, until synchronized spontaneouslybeating cell monolayers were formed, than maintained in serum freeculture media (contained Insulin, BSA, and Vitamin B₁₂) preconditionedon cultured fibroblasts.

AAV Transduction

Transfection experiments with serial virus titers were performed twice.

During the first experiment, transfections of myocytes in MEA chamberswere carried out on 5th day in culture at the following titers: 10¹¹,10¹⁰, 10⁹, 10⁸ vg/ml of B-AAV1-HCN4st1 and non transduction negativecontrol (5 arrays for each condition).

During the second experiment, in addition to B-AAV1-HCN4st1 and negativecontrol, a control of B-AAV1-empty particles was included.

MEA System

The day before transduction, and then each following day, electrogramsfor each culture were recorded with MEA system and microscopicfluorescence and bright field images of each array were taken with Leicainverted microscope at 10× magnification. Cultures were monitored forabout 2 weeks, after which MEA recordings were processed using “MC Rack”Multi Channel Systems Software and analyzed in Microsoft Excel;microscopic images were processed with “ImageJ” software.

NRVM cultures showing signs of degradation were rapidly assessed forapoptosis with Vibrant Apoptosis Assay Kit #5 (Invitrogen, Carlsbbad,Calif., USA) based on fluorescence detection of the compacted state ofthe chromatin in apoptotic cells.

Results

HCN4st1, st2 and st3 evaluation by patch clamp.

We have expressed three HCN4st constructs individually in HEK293 cellsby Fugene 6 transfection and performed whole-cell patch clampexperiments to assess their electrophysiology function. Our experimentssuggested that only HCN4st1 remained indistinguishable from the HCN4t,thus suggesting that truncation at N-terminus of 213 amino acids doesnot compromise HCN4 function (FIG. 8). When using HEK293 cells HCN4st2and HCN4st3 did not lead to functional HCN4 current, indicating thatthese constructs may not function properly in HEK293 cells, or thatfurther modification on these two deletions may be needed. HCN4st1 genecontains only 1575 by nucleotides, well within the packaging capacity ofscAAV vector system, and has both the C-terminus and N-terminustruncated.

B-AAV1-HCN4st1 In Vitro Evaluation by MEA

Non transduced control NRVM cells demonstrated stable (˜20-40 BPM)beating rates and overall uniform healthy appearance during the two weekobservation period (FIG. 9).

NRVM cells transduced with 10¹¹ vg/ml of B-AAV1-HCN4st1 were not beatingby the next day after transduction and rapidly degraded showing thesigns of apoptosis (FIG. 10).

For the next lower titer −10¹⁰ vg/ml NRVM cells reached the maximum ofbeating rate ˜115 BPM by day 3 after transduction, and then degraded byday 7.

For the 10⁹ vg/ml titer, NRVM cells reached maximum of beating rate˜130-160 BPM by the day 6 and degraded by the day 10.

For the lowest tested 10⁸ vg/ml titer, NRVM cells reached beating rate˜75 BPM by the day 10 and did not show signs of degradation (FIG. 11,FIG. 12).

NRVM cells transduced with B-AAV1-empty particles did not show any signsof degradation and any noticeable rate increase compared to nontransduced controls (FIG. 13).

Overall, neonatal rat ventricular myocytes transduced withB-AAV1-HCN4st1 showed increased induced beating. At higher titers (10¹¹,10¹⁰, 10⁹ vg/ml), B-AAV1-HCN4st1 caused cell degradation via apoptosis.At lower titer (10⁸ vg/ml), B-AAV1-HCN4st1 was safe and reached inducedbeating rate at ˜75 BPM.

Example 2

AAV1-HCN4-mediated biological pacemaker paces the canine heart with AVblock over 7 months and responds well to autonomic challenges

Implantation of an electronic pacemaker is necessary for a patient withsevere bradycardia; however, while effectively improving the lives ofmany patients, such therapy has several limitations including hardwarecomplications, limited battery life and lack of response to autonomicand physiologic demands on hearts. Compared to its counterpart, abiological pacemaker is a conceptually attractive alternative toelectronic pacemakers.

This study was to test whether using adeno-associate virus-1 (AAV1) withtruncated human HCN4 (hHCN4tr) would create a long-term (>6 months) andsustained (nearly 100% biologically induced pacing) biological pacemakerin the left ventricle (LV) of canines with atrioventricular (AV) nodalblock.

Canines (n=3) with neutralizing anti-AAV 1 antibodies (NAb) at a titer≦1:40 serum dilution were selected. After complete AV block, theAAV1-hHCN4tr vectors were epicardially injected into the LV apex using aside-hole needle as described in Hiniduma-Lokuge et al. (PCT PublicationNo. WO/2008/055001). An electrical pacemaker (VVI 50 bpm) was implantedfor backup pacing and recording of the electrocardiograms every twohours. During the follow-up monitoring, drug and exercise challengeswere performed. Twenty-four hour Holter monitoring and pacemaker logrecord checks were also performed.

All canines demonstrated biological pacemaker activities 3 days afterAAV1-hHCN4tr injection. The biological pacemaker in two canines with NAbat a titer of 1:40 diminished 3 weeks after the gene transfer presumablydue to the pre-existing immunity to AAV1. However, the canine with NAbat a titer of 1:20 has exhibited sustained biological pacing activitiesat a range of 60-150 bpm over 7 months at the time of this abstractsubmission (FIG. 15). The electrical pacemaker intervened at 50 bpm whenspontaneous ventricular rate fell below at that rate, triggering for <2%of the beats. The biological pacemaker responded well to drugs, such asisoproterenol and metoprolol, and also showed physiological diurnalvariations and responses to exercise.

CONCLUSION

LV epicardial injection with AAV1-hHCN4tr creates long-term (over 7months) and sustained (nearly 100% biologically induced pacing)biological pacemaker activities in a complete AV block canine model.Interestingly, such biological pacemaker responds well to autonomicchallenges and physiological exercise.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A method comprising: identifying a distal end of a catheter at a tissue site of a patient; delivering a fluid or polymer that contains an HCN polynucleotide comprising a nucleotide sequence encoding an HCN polypeptide having channel activity, wherein the amino acid sequence of the HCN polypeptide and the amino acid sequence of a reference polypeptide have at least 80% identity, the reference polypeptide beginning with an amino acid selected from amino acids 92-214 and ending with an amino acid selected from amino acids 723-1188 of SEQ ID NO:8 to the tissue site of the patient via the catheter.
 2. The method of claim 1 wherein the identifying comprises electrically sensing contact between a distal end of a catheter and the tissue site of the patient, and the method further comprises delivering an electrical stimulus to the cardiac tissue site of the patient to enhance transfer HCN polynucleotide to the tissue site via electroporation.
 3. The method of claim 2 wherein delivering the electrical stimulus to the tissue site includes delivering the electrical stimulus to the tissue site via the catheter.
 4. The method of claim 2 wherein delivering the electrical stimulus to the tissue site via the catheter comprises delivering the electrical stimulus to the tissue site via an electrode coupled to the catheter and an electrode coupled to a distal tip of a probe extending from the catheter.
 5. The method of claim 2 wherein delivering the electrical stimulus to the tissue site via the catheter comprises delivering the electrical stimulus to the tissue site via an electrode coupled to the catheter and a distal tip of a probe extending from the catheter, the distal tip of the probe being formed from an electrically conductive material.
 6. The method of claim 2 wherein delivering the electrical stimulus to the tissue site includes delivering the electrical stimulus to the tissue site via an implanted medical device.
 7. The method of claim 2 wherein the electrical stimulus delivered to the tissue site comprises a stimulation pulse.
 8. The method of claim 2 wherein the electrical stimulus delivered to the tissue site comprises a series of stimulation pulses.
 9. The method of claim 2 wherein delivering fluid to the tissue site of the patient via the catheter includes delivering fluid to the tissue site of the patient via one or more exit ports of a distal tip of a probe extending from the catheter.
 10. The method of claim 9 wherein the distal tip of the probe comprises a needle.
 11. The method of claim 9 wherein the distal tip of the probe comprises a helix shaped distal tip.
 12. The method of claim 9 wherein the distal tip of the probe extends from a body of the catheter upon sensing contact between the tissue site of the patient and the catheter.
 13. The method of claim 1 wherein the HCN polynucleotide is present in a vector.
 14. The method of claim 13 wherein the vector is a viral vector, a transposon vector, or a plasmid vector.
 15. The method of claim 14 wherein the viral vector is a single strand adeno-associated virus or a self complementary adeno-associated virus.
 16. A system comprising: a fluid supply; a catheter that includes a catheter body that defines an inner lumen, a probe within the inner lumen that delivers fluid from the fluid supply to a tissue site of a patient, and at least one electrode coupled to the catheter to detect contact between the catheter and the tissue site, wherein the fluid comprises an HCN polynucleotide of claim 1; and a power supply to generate an electrical stimulus that is delivered to the tissue site.
 17. The system of claim 16 further comprising a pump to drive fluid from the fluid supply through the catheter.
 18. The system of claim 16 wherein the power supply comprises an implanted medical device that delivers the electrical stimulus to the tissue site.
 19. The system of claim 18 wherein the implanted medical device comprises one of an implantable pulse generator, an implantable cardioverter/defibrillator, and an implantable pacemaker/cardioverter/defibrillator.
 20. The system of claim 16 wherein the fluid supply comprises an implanted fluid reservoir.
 21. The system of claim 16 wherein the power supply is coupled to the catheter, and the catheter delivers the electrical stimulus to the tissue site.
 22. The system of claim 21 wherein the probe includes a distal tip made from an electrically conductive material and the electrode is coupled to the catheter body, and the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the distal tip of the probe.
 23. The system of claim 21 wherein the catheter includes a pair of electrodes, a first electrode coupled to the probe and a second electrode coupled to the catheter body, and the catheter delivers the electrical stimulus to the tissue site via the electrode coupled to the catheter body and the electrode coupled to the probe.
 24. The system of claim 16 wherein the electrical stimulus delivered to the tissue site includes a stimulation pulse.
 25. The system of claim 16 wherein the electrical stimulus delivered to the tissue site includes a series of stimulation pulses.
 26. The system of claim 16 wherein the fluid comprises a polymer. 